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Platelet dysfunction may be inherited or acquired. In this study platelet dysfunction was assessed in both contexts; the inherited platelet disorder ACTN1-related Congenital Macrothrombocytopenia (CMTP) and the acquired prothrombotic state in Multiple Myeloma (MM). Actinin-1 is a widely-expressed actin binding protein but mutations have a tissue-specific impact, causing CMTP, characterised by low platelet count and large platelet size. It was previously shown that mutations in the actin-binding domain of actinin-1 increase the affinity of actinin-1 for actin filaments. In this study, nine mutations in rod domain and calcium-binding CaM-like domain were assessed in terms of their impact on actin binding and calcium binding. It was shown that calcium binding was not significantly affected by mutations in the CaM-like domain, although thermal stability was slightly decreased, and mutations in both the rod and CaM-like domain increased actinin's ability to bundle actin filaments in-vitro. Thus, CMTP-causing actinin-1 mutations outside the actin-binding domain also increase actin association, suggesting a common molecular mechanism underlying actinin-1 related CMTP. The tissue specific functions of actinin-1 in platelet production were explored. Megakaryocyte maturation in terms of polyploidy was assessed in CMK and MEG-01 cell lines expressing mutant actinin-1 but no significant disruption was noted. Primary murine megakaryocytes were used to image WT actinin-1 and actinin-4 in two key structural features of platelet production; podosomes and proplatelets. It was shown that more actinin-1 was expressed in platelets and megakaryocytic cell lines than actnin-4, possibly explaining the tissue-specific impact of actinin-1 mutations. The presence of actinin-1/4 heterodimers has not been explored in these cells and a method to assess actinin-1/actinin-4 heterodimers was optimised. It was also shown that integrin αIIβ3 interacts with actinin-4 or actinin-1/4 heterodimers, implying that this is not an actinin-1 specific interaction. Consideration of actinin isoform specific functions is of value in future study to determine the role actinin-1 plays in platelet production. In the second part of this project, platelet activation status was assessed in myeloma patients. Thrombotic events are common in patients with multiple myeloma (MM), smouldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS). In the present study, multiparameter analysis of platelet activation and responsiveness was investigated by flow cytometry in patients with MGUS, SM/MM and healthy controls (HCs). The median platelet surface levels of CD63, annexin V and PAC-1 antibody (specific for activated integrin αIIbβ3) binding were significantly elevated in patients with MGUS versus the HCs. In all, 74% of MGUS and 38% of SM/MM patients had one or more elevated marker of platelet activation, compared to 19% of the HCs. Marker-specific hyporesponsiveness of platelets to agonist [adenosine diphosphate (ADP), thrombin receptor-activating peptide 6] stimulation in-vitro was observed, with significantly reduced surface levels of P-selectin in response to ADP in patients with MGUS. Platelet-associated immunoglobulins were elevated in a subset of patients. A subset of patients were also analysed after two months of anti-myeloma treatment and anti-thrombotic therapies where prescribed. The hyperactive, hyporesponsive state was not significantly altered at this timepoint post diagnosis. However, a significant elevation in reticulated platelets and platelet-leucocyte aggregates was found. As risk assessment models evolve to include novel laboratory parameters, these observations suggest that further investigation of the predictive and prognostic value of platelet activation markers in such patients is warranted. Overall this project contributes to what is known about actinin-1 related CMTP, as mutations outside the actin binding domain appear to also increase actinin's ability to bundle actin filaments. The impact of this gain-of-function disruption remains to be determined and several features of platelet production which involve actinin-1 are highlighted for further study in this project. Platelet dysregulation was demonstrated as a feature of MM, SM and pre-malignant MGUS and could be explored as a potential indicator of thrombotic risk in these patients in future study. |
